Expression of RUNX1-RUNX1T1 Alone Has No Effect on the Intrinsic Susceptibility to Cytotoxic Chemicals.

Abstract The RUNX1 gene (aka AML1 on chromosome 21) encodes the alpha component of the Core Binding Factor (CBF) complex. This heterodimeric transcription factor is central to haematopoietic development and has been shown to be involved in the regulation of a number of haematopoietic-specific genes including IL-3, MPO and GM-CSF. RUNX1 is one of the most frequently disrupted genes in acute myeloid leukaemia (AML); and is particularly associated with chromosomal translocations. The t(8;21) encodes the RUNX1-RUNX1T1 (aka AML1-ETO) fusion protein which promotes self-renewal of haematopoietic cells and also inhibits their subsequent differentiation. Leukaemias expressing this abnormality are generally associated with a good prognosis in terms of complete remission, relapse risk and overall survival compared with other subtypes and tends to respond favourably to chemotherapeutic agents. However it is not currently known why patients expressing the t(8;21) have a good prognosis. It has been suggested that in AML patients expressing RUNX1-RUNX1T1, the fusion gene may promote the expression of p-glycoprotein (encoded by MDR-1) in mediating drug resistance. We therefore tested this hypothesis directly by expressing the RUNX1-RUNX1T1 fusion as a single abnormality in human haematopoietic cell subsets and performed Affymetrix microarray analysis to determine whether this fusion had any effect on the transcription of MDR genes. Using this approach we generated independent replicate sets of data from control and RUNX1-RUNX1T1 matched CD34+ cultures as well as matched sets constituting granulocytic (CD14lo, CD36lo, CD15hi) and monocytic (CD14hi) unilineage populations (isolated from day 6 cultures by immunomagnetic sorting). cRNA was prepared from each sample and hybridised to Affymetrix human 133A oligonucleotide arrays which allowed the simultaneous analysis of 6 MDR family gene members. In each of these populations, the expression of MDR genes was not significantly different from controls. We could therefore find no evidence that RUNX1-RUNX1T1 expression directly influences MDR gene expression as a single abnormality. We next addressed the issue of whether the t(8;21) abnormality directly influences the susceptibility to chemotherapeutic agents. We therefore assessed the sensitivity of CD34+ cells expressing RUNX1-RUNX1T1 to a number drugs commonly used to treat AML (Daunorubicin, Cytarabine, Fludarabine, Idarubicin or Etoposide) in comparison with matched controls. Remarkably, none of these agents differentially affected the growth of RUNX1-RUNX1T1 transduced cells. Since treatment of AML commonly involves multiple drugs, we also determined the effect of combining two or more of these chemotherapeutic agents. Again, we observed little difference in the in vitro growth response of RUNX1-RUNX1T1 expressing cells compared to controls. Taken together, these data suggest that expression of RUNX1-RUNX1T1 itself has no effect on the intrinsic susceptibility to cytotoxic chemicals. This raises the alternative hypothesis that RUNX1-RUNX1T1 moderates the influence of secondary abnormalities which are required for RUNX1-RUNX1T1 expressing cells to undergo leukaemic transformation.